Efficient Bioproduction of Human Milk Alpha-Lactalbumin in Komagataella phaffii .

Alpha-lactalbumin (α-LA; the most abundant whey protein in human milk) contributes to infant development, providing bioactive peptides and essential amino acids. Here, Komagataella phaffii ( K. phaffii ) was selected as the production host. We found that the K. phaffii host X33 was suitable for expressing the target protein, yielding 5.2 mg·L -1 α-LA. Thereafter, several secretory signal peptides were applied to obtain a higher titer of α-LA. The strain with α-factor secretory signal peptide secreted the highest extracellular titer. Additionally, promoters AOX1 , GAP , and GAP(m) were compared and applied. The strain with the promoter AOX1 produced the highest extracellular titer. In addition, coexpressing human protein disulfide isomerase A3 (h PDIA3 ) increased the titer by 27%. Human α-LA production by the strain X33-pPICZαA-hLALBA-hPDIA3 reached 56.3 mg·L -1 in a 3 L bioreactor. This is the first report of successful secretory human α-LA expression in K. phaffii and lays foundations for the simulation of human milk for infant formulas and further development of bioengineered milk.